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Botany Plant Molecular Biology Chromosomes
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Subject Name <BOTANY>
Paper Name <Plant Molecular Biology>
Module Name/Title <Chromosomes>
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Pre-requisites
Objectives To make the students aware of the discovery, structure and function of chromosomes
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<Chromosomes > <Sub-topic Name1>, <Sub-topic Name2>
<Topic name2> <Sub-topic Name2.1>, <Sub-topic Name2.2>
Botany Plant Molecular Biology Chromosomes
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Botany Plant Molecular Biology Chromosomes
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2.2.1 Historical Perspectives
Chromosomes, the carriers of hereditary material, are the thread-like organized aggregates of nucleic acids and proteins present in the cells of all organisms. These remain dispersed throughout the major part of cell cycle and thus, cannot be resolved by light microscopy. However, chromosomes become conspicuous at metaphase stage of cell division in their most condensed form. Chromosomes were noted by many famous scientists during the second half of nineteenth century as thread or rod-like structure which come to lie between two poles during each cell division. Walther Flemming (figure 2), a German anatomist who coined the terms chromatin and mitosis and described the behavior of chromosomes during mitosis including their splitting along the length and distribution of the two to the daughter cells, was the first to describe chromosomes in his book entitled, “Zell-substanz, Kern und Zelltheilung” (“Cell-Substance, Nucleus, and Cell- Division”) published in 1882. Flemming noted that the fibrous material that was termed as chromatin by him condenses into thread like stainable structures which he termed as
‘mitosen’, meaning threads in Greek. However, Eduard Strasburger, (Fig. 3) who coined the terms cytoplasm and nucleoplasm and was first to describe cell division in a plant cell, too noted chromosomes without naming them. The term ‘chromosome’ was coined by German anatomist, physiologist and pathologist Wilhelm Waldeyer (Fig 1) in 1888 for the structures which become evident during cell division and take up stain with basic dyes.
Prior to him, Boveri had described these structures as “chromatic element” and Flemming named them as ‘mitosen’. The term chromosome is derived from Greek words where chromo=color and soma=body.
Botany Plant Molecular Biology Chromosomes
Fig. 1: W. Waldeyer Fig. 2: W. Flemming Fig. 3: E. Strasburger Fig. 4 T. Boveri Fig. 5: W. Sutton
2.2.1.1 Chromosomal Theory of Inheritance
Though, at the time of Christianizing of chromosomes it was known that these elements are involved in cell division, their significance in transmission of genetic characters was not realized till 1902. By this time Mendel’s laws were re-discovered. In 1902, German scientist Theodor Boveri (fig. 4) and American, Walter Sutton (Fig. 5) independently suggested that chromosomes could be the carriers of Mendel,s independently segregating factors or the genes as we call them now. This came to be known as Chromosomal Theory of Inheritance. In fact, the observation of Flemming that chromosomes split along their length during mitosis and partition in the daughter cells paved way for linking of chromosomes with hereditary mechanisms. Boveri based on his observations on the early development of Ascaris megalocephalo possessing only two pairs of chromosomes which could be easily studied in colorless cells of embryos, concluded that (i) germ cell lineage provides continuity between generations, (ii) the number of chromosomes in Ascaris eggs is reduced to half (2) and full complement (4) of chromosomes is restored after fusion with sperm and (iii) the full complement of chromosomes is retained only in Ascaris germ cell lineage. Further, based on a series of experiments on sea urchin eggs, Boveri demonstrated that each chromosome affected the development from sea urchin eggs. These eggs can be fertilized by two sperms and the resulting zygotes can have variable number of chromosomes. Only the embryos with complete set of 36 chromosomes develop normally. Thus, Boveri in 1902 opined that a specific assortment of chromosomes was responsible for normal development implying that individual chromosomes possess different qualities. In his seminal papers, Boveri also emphasized that principles of segregation and independent assortment enunciated by Mendel operate at cellular level as the characters dealt with Mendel’s experiments are
Botany Plant Molecular Biology Chromosomes
connected to specific chromosomes, indirectly implying that chromosomes are the carriers of ‘factors’ (presently genes) that are responsible for these factors.
Walter Sutton who was an American graduate student extensively studied meiosis in testes of grasshopper, Brachystola magna. Sutton could recognize individually all the chromosomes (11 pairs + a sex chromosome) of the organisms based on their morphology and could follow the behavior of each including the sex chromosome during all stages of meiosis. Based on these studies, he concluded in 1902 that the physical basis of heredity as proposed by Mendel could be observed association of paternal and maternal chromosomes during meiosis and their segregation at a later stage. He also noted that the segregation of individual chromosomes to the daughter cells is independent of other chromosomes. As a result, the resultant gametes receive a random mix of chromosomes from maternal and paternal sides. The number of possible combinations of the chromosome complements which the gametes could acquire is 2n, where n is the number of chromosomes. The fusion of the two gametes leading to the formation of zygote doubles the possible combinations to 2x2n. According to Sutton, this could be the basis of variation in heritable traits. Thus, Sutton not only confirmed the observations of Boveri but also extended these.
By the beginning of twentieth century, chromosomes, noted only as cytological entities during the second half of the preceding century, had not only acquired their name but were also established as genetical entities. If one has to give a present day definition to chromosomes, these can be considered as assemblies of transcriptional units, interspersed with regulatory sequences and associated proteins, which are precisely replicated during each cell division. Though, functionally chromosomes in all organisms are similar, do chromosomes all organisms share a common structural organization? The answer to this question is provided briefly in the next section and the details would be provided in subsequent modules of the unit.
2.2.2 Types of Chromosomes
Though chromosomes of all organisms are made up of nucleic acids and proteins, how these constituents associate to give rise chromosomes exhibit some broad variations, on the basis of which chromosomes can be divided into three categories. For the sake of
Botany Plant Molecular Biology Chromosomes
brevity, these can described as (i) prokaryotic chromosomes, (ii) mesokaryotic chromosomes and (iii) eukaryotic chromosomes.
2.2.2.1 Prokaryotic Chromosomes
As the name indicates, these chromosomes are found in prokaryotes. Such chromosomes are neither nucleosomal, nor enclosed in defined a compartment. The traditional views that a prokaryotic cell contains a single circular DNA molecule, aggregated as a nucleoid lying in the cytoplasm, have undergone a sea change. These chromosomes though not having nucleosomal configuration are associated with proteins which are histone-like. In all planctomycetes, a group of peptidoglycan-less bacteria forming a distinct phylum Planctomycetes, nucleoid is surrounded by a membrane.
Moreover, though most of the studied prokaryotes including Escherichia coli have a single circular chromosome, there are now many examples of prokaryotes having multiple chromosomes, some of which could be linear. First bacterium shown to possess linear chromosome was Borrelia burgdorferi
,
the causal agent of Lyme disease. This was revealed by pulse-chase agarose gel electrophoresis, a technique described by Schwartz and Cantor in 1984, where chromosome of Borrelia behaved as a eukaryotic linear chromosome comprising about 1000 bp. In addition, this species also contains many circular and linear plasmid which vary in size from 10 -60kb. The photosynthetic bacterium, Rhodobacterium sphaeroides, was the first to be shown to possess two chromosomes both of which are circular. Agrobacterium tumefaciens has four chromosomes, one of which is linear. Another example of a bacterium having more than one chromosome is Sinorhizobium meliloti having three circular chromosomes. Till 2009, of the 799 bacteria whose complete genome was available, 70 possessed secondary chromosomes. A new term ‘chromid’ has been proposed for these accessory chromosomes of bacteria because of their plasmid type replication and maintenance systems, though possessing nucleotide composition similar to that of chromosome and carrying core genes found in chromosomes of other species. The detailed organization of prokaryotic chromosomes would be discussed in a subsequent module.Botany Plant Molecular Biology Chromosomes
2.2.2.2 Mesokaryotic Chromosomes
Mesokaryote, a term used by Dodge in 1965 for dinoflagellates (fig. 6, 7), a diverse group of unicellular protist algae, exhibiting unusual nuclear features. As the term (meso:
in between and karyon: nucleus) indicates these organisms share features with both prokaryotes and eukaryotes. Like eukaryotes, dinoflagellaes possess organelles including a well-defined nucleus having a typical eukaryotic nuclear envelope that encloses multiple chromosomes, which remain condensed throughout the cell cycle. However, these chromosomes lack histones and do not exhibit nucleosomal configuration. The nuclear division in these organisms too shares features with both prokaryotes and eukaryotes as nuclear envelope persists during division. An extranuclear spindle, to which chromosomes are attached through nuclear envelope, is also formed. Therefore, these chromosomes have been termed as mesokaryotic. We will learn more about these chromosomes in a subsequent module along with those of prokaryotes.
Fig. 6: A typical dinokaryon in 'Gymnodinium' sp. Fig. 7: Schematic drawing of a generalized motile dinoflagellate cell with theca showing ultrastructural characteristics.
(Fig. 5 and 6 are from: http://tolweb.org/Dinoflagellates/2445)
2.2.2.3 Eukaryotic Chromosomes
Third and the most extensively studied type of chromosomes are those contained by eukaryotes. These chromosomes are nucleosomal in configuration, always linear and
Botany Plant Molecular Biology Chromosomes
are contained in a typical eukaryotic nucleus surrounded by a double membrane nuclear envelope. Eukaryotes have multiple chromosomes, each containing a single molecule of DNA. Eukaryotic chromosomes because of their complex nature have been studied extensively for their molecular architecture (anatomy) and its effect on their behavior and functions. In this unit we will be studying different aspects of eukaryotic chromosomes and therefore, the term chromosome used subsequently in following account in this module or in subsequent ones, except that on prokaryotic and mesokaryotic chromosomes, refers to only eukaryotic chromosomes.
2.2.3 Structure of a Metaphase Chromosome
For predominant part of the cell cycle, chromosomes remain in extended and dispersed form and therefore, cannot be visualized. It is during the metaphase stage of mitosis meiosis that chromosomes exist in their most condensed state. By this stage, chromosome DNA has duplicated and the resultant/daughter chromosomes referred to as sister chromatids can be seen attached to each other at a point known as centromere. In a single separated chromosome this appears as a constriction. Thus, centromere divides chromosome in two parts, generally known as arm, with the shorter traditionally referred to as ‘p’ arm and longer as ‘q’ arm (Fig. 8). The physical ends of the chromosomes are known as telomeres.
Botany Plant Molecular Biology Chromosomes
Fig. 8: A metaphase chromosome as seen under a electron microscope (right), diagrammatic representation of the same (p: short arm and q: long arm of the chromosome)
(http://www.elu.sgul.ac.uk/rehash/guest/scorm/53/package/content/metaphase.htm).
Centromeres, having a complex structure, are composed of repetitive sequences and specific proteins. Besides holding the sister chromatids together at meiosis I and metaphase stage of mitosis, centromeres ensure the equal segregation of chromosomes to the daughter cells. This is facilitated by the movement of two daughter chromosomes to two opposite poles through spindle fibers attached to centromeres through multiprotein structures named as kinetochore on the centromere. Telomeres, the ends of chromosomes, are also generally composed of repetitive sequences with specific proteins.
Telomeres are multifunctional units. Telomeres (i) prevent end to end joining of chromosomes (making them non-sticky), (ii) ensure maintenance of length of chromosomes by preventing loss of DNA from the end of chromosomes following DNA replication and (iii) may also help in positioning of the chromosomes within a nucleus.
Botany Plant Molecular Biology Chromosomes
2.2.4 Euchromatin and Heterochromatin
With the completion of mitosis chromosomes start uncoiling. However, certain sections of chromosomes remain dark staining even at interphase stage. These darkly staining regions of the interphase nuclei are called heterochromatin and the areas where chromatin takes less stains are called euchromatin. Likewise, even the condensed chromosomes have structurally and functionally different euchromatic and heterochromatic regions. As heterochromatin remains condensed throughout the life of a cell it is gene- poor, contains hugely repeated sequences of DNA or repressed genes and consequently, is transcriptionally inactive. Euchromatin on the other hand is less condensed. This region is gene-rich and transcriptionally active. Most frequently, heterochromatin is associated with centromere and telomere of the chromosome. However, it should not be implied that all the inactive genes or non-transcribed regions in a chromosome are visible as heterochromatin, nor it is necessary that regions visible as heterochromotin are never transcribed. Rather, in many cases these are interchangeable states and provide an effective method of gene regulation, the molecular mechanisms and the functional implications of heterochrtomatization will be discussed in the chapter on gene regulation.
However, over here we will discuss some of classical examples of heterochromatization leading to gene activation. The best example of heterochromatization leading to gene inactivation is that of one of the X chromosomes in the somatic cells of all female mammals. Early in the embryonic development, one of the X chromosomes, either from the maternal (Xm) or paternal (Xp) side gets inactivated randomly. Thus, in some cells Xm is inactivated, while in others Xp gets inactivated. The progeny cells derived from these cells continue to
Botany Plant Molecular Biology Chromosomes
Fig. 9: Murray L. Barr Fig. 10: Mary Lyon
(http://www.cmaj.ca/site/ (https://www.mtholyoke.edu sites/default/
100/graphics/barrLrg.jpg) files/styles/175_women_gallery_ feature/
public/175/images/lyon_mary_chandler _painting.jpg?itok=D0jMSkSI)
have same condition. As a result, body of an adult mammalian female becomes genetically mosaic. The inactivated X chromosome can be seen as a dense nuclear structure, termed as Barr body, named so after the British cytologist, M. L. Barr (fig. 9).
Genetic consequence of this inactivation was highlighted by Mary Lyon (Fig. 10) in 1961 to explain the mottled phenotype of female mice heterozygous for specific coat color mutations. A similar mechanism is responsible for mosaic coat colour of tortoise shell cats. In cats, one of the genes controlling coat colour is located at X chromosome. This gene has two alleles, one coding for white (o) hairs and other yellow (o), the latter being dominant over the former. Males would have two genotypes, which are XoY and XoY, consequently the coat colour among males would be either white or yellow. Among female cats three possible genotypes are XoXo, XoXo and XoXo. Cats with the first two genotypes would have white or yellow colour coats, respectively. The last genotype should result in yellow colour coat cats as the yellow is dominant. However, this genotype produces cats with coats having patches of yellow and white because of random inactivation of one of the X chromosome in different cells at an early stage of development.
Thus, cell derived from a cell in which Xo has been inactivated would produce patches
Botany Plant Molecular Biology Chromosomes
with white fur, while those derived from cell having inactivated Xo contribute to the formation of yellow patches (Fig. 11).
Fig. 11: Schematic representation to explain genetical basis of different coat colours in tortoise shell cats (http://www.sciencebuddies.org/Files/3344/5/MamBio_img039.jpg)
To distinguish between the regions which remain permanently condensed from those which can exist in both states the terms obligate heterochromatin for the former and facultative heterochromatin for the latter are used.
2.2.4 Special Staining Methods for Chromosomes
Till 1960s, traditional way of studying the chromosomes at metaphase stage was to stain them with classical cytological stains, such as, aceto-orcein, acetocarmine, gentian violet and haemotoxylin, and then to identify individual chromosome on the basis of position of centromere or secondary constriction. However, in many cases chromosomes become indistinguishable because morphologically they happen to be identical. In late 1960s, the foundation for the subsequent development of chromosome banding was laid by Caspersson, who predicted that because of the differences in base composition along the length of chromosome, DNA-binding dyes might produce different intensities along the length of the chromosome. This was demonstrated to be true when in 1968 fluorescent banding was demonstrated in plant chromosomes stained with fluorescent dye quinacrine
Botany Plant Molecular Biology Chromosomes
and in 1971 the technique was replicated to produce banding patterns for all 24 human chromosomes (Fig. 12). This came to be known as Q-banding and was followed by development of other banding methods. All these have been described below briefly.
2.2.4.1 Q-banding
As already described, a fluorescent dye, like quinacrine, is used for staining the chromosomes. The regions of chromosome that are rich in the bases adenine (A) and thiamine (T) fluoresce more intensely than those having abundance of guanine (G) and cytosine (C). As the fluorescence does not last for more than few minutes, these preparations have to be photographed as early as possible. Besides, quinacrine, flurescent stains, Hoescht 33258, DAPI (4’,6’-diamino-2-phenylindole) and DIPI (diimidazolinophenylindole) also produce Q-bands similar to those by quinacrine.
Fig. 12: Q-banded human chromosomes (http://www.mun.ca/biology/scarr/Fig17_05a.html)
2.2.4.2 G-banding
G-banding method, introduced soon after Q-banding, makes use of Giemsa stain and is therefore, known as G-banding. The advantage over Q-banding is that it yields permanent stain and can be observed and photographed under a standard light microscope. The procedure involves pre-treatment of chromosome with trypsin, followed
Botany Plant Molecular Biology Chromosomes
by staining with Giemsa. Giemsa stained portions of chromosomes match with bright bands produced by Q-banding (Fig. 13).
Fig. 13: G-banded human chromosomes
(http://www.biologyreference.com/images/biol_01_img0082.jpg)
2.2.4.3 R-banding
R in R-bandining refers to reverse banding. This method of chromosome banding has been named so because the intense bands produced are opposite to those produced by Q-banding. The reason is that the stains used have more affinity to GC-rich regions than AT-rich portions. The fluorescent R-banding patterns are produced by dyes, chromomycin A3, olivomycin and methramycin. R-bands also develop if the chromosomes are subjected to high temperature (rather than trypsin as in G-banding) for several minutes before stained with Giemsa stain.
2.2.4.4 C-banding
Such type of bands develop only in constitutive heterochromatic regions of chromosomes, i.e. centromere etc. The chromosomes are extracted for nucleic acids and proteins by treating with acidic and basic solutions followed by staining with Giemsa stain.
Most of the chromatin in euchromatin reions is extracted because of their less condensed nature, while heterochromatic regions are relatively less affected. Therefore, when treated
Botany Plant Molecular Biology Chromosomes
chromosomes are stained with Giemsa or any other DNA specific stain heterochromatic regions are stained more intensely than euchromatic regions (fig. 14).
Fig. 14: C-banded chromosomes of fish, Notothenia rosii (http://www.acanthoweb.fr/sites/default/files/Notothenia- rossii_marquage_des_chromosomes_en_bandes_C.jpg)
Besides above-described staining methods, a number of other staining procedures for high resolution banding and identifying specific coding or non-coding portions of the chromosomes have been developed in recent times. However, it is beyond the scope of this module to describe all these which would be discussed in other modules dealing with cytogenetics.
2.2.6 Unineme Model of Chromosomes
How many DNA molecules are present in each chromosome or chromatid of divided chromosome at the metaphase stage? Are there more than one DNA molecule arranged parallel or end to end in each chromosome (multineme model) or only one DNA molecule run from one end to the other in each chromosome (unineme model)? These questions had intrigued many because it was not possible to visualize individual chromosomes at interphase stage or DNA molecule(s) present in each highly condensed chromosomes at metaphase stage. While describing the characteristic features of eukaryotic chromosome it was emphasized that each of these contains a single DNA molecule, implying that unineme model of chromosome is the accepted one. Let us see
Botany Plant Molecular Biology Chromosomes
the experimental evidences which support the conclusion that each chromosome contains a single DNA molecule that runs from one end to the other through centromere.
2.2.3.1 Biochemical and Ultrastructural Studies on ‘Lampbrush’ Chromosome
One piece of evidence supporting the unineme nature of chromosomes emanated through the studies on “lampbrush chromosomes” (fig. 15). These chromosomes which are several hundred µm micrometers in length can be visualized at prophase I stage of oogenesis in many vertebrates including amphibians. At this stage, each lampbrush chromosome comprises two chromatids with central condensed axis from which many radial loops emanate on both sides (Fig. 7). The radial loops show high transcriptional activity.
Fig. 15: A portion of Lampbrush chromosome showing a
central axis and radial loops
(http://www.cbs.dtu.dk/staff/dave/roanoke/fg16_0.jpg)
The treatment with DNAase breaks loops as well as the central axis in fragments, while treatments with RNAase and/or proteins, though removes matrix material, the integrity of matrix and loops remains intact. The chromosomes treated with RNAase and proteases when ‘viewed’ through electron microscopy revealed 20 nm thick central filament in each loop, equal to thickness of a DNA double helix. As each lampbrush chromosomes comprises two paired chromatids with the filaments in each loop having thickness equal to that of DNA duplex, it can be concluded that each chromatid represents a DNA molecule associated with proteins and RNA. The nuclease digestion kinetics of the loops in lampbrush chromosomes too yielded results expected of a DNA double helix.
Botany Plant Molecular Biology Chromosomes
2.2.3.2 Sedimentation Analysis of Drosophila Chromosomes
One way of ascertaining the unineme nature of the chromosome is through an experiment that involves isolation of DNA from nuclei of an organism under conditions of least shearing. The mixture of DNA molecules so isolated can be separated by density gradient centrifugation. Based on the sedimentation coefficient of DNA molecules in each band, mass of each can be calculated. Simultaneously, the chromosomes of the same organism can be stained with a DNA specific stain and the amount of DNA present in the largest chromosome can be estimated using microspectrophotometry. Based on this atomic mass of the DNA molecule present in this chromosome can be calculated. If the two figures obtained so match, one can safely conclude that a single molecule of DNA is present in a chromosome. It is not possible to isolate intact DNA molecules from human chromosomes because of the large size; the average length of DNA present in each of human chromosome is about 5 cm. However, in lower eukaryotes like Drosophila, this experiment has been performed with a variety of genetically different forms with similar results.
2.2.3.3 Viscoelastmetric Studies of Drosophila Chromosomes
A direct way of demonstrating the unineme nature of chromosomes could be to isolate DNA from all chromosomes of an organism followed by their separation by an analytical method to find out the number of DNA molecules equal to the number of chromosomes and the calculated molecular weight of each equal to the estimated amount of DNA in each chromosomes by mirospecrophotometry. However, this is generally not possible because of very high length to width (diameter) ratio of a DNA molecules, making it highly sensitive to shearing during a biochemical procedure. An alternative method that has been used to provide evidence for the presence of single giant DNA molecule in a chromosome is viscoelastometry. In simple words, the technique involves stretching a molecule in a solution by applying a driving force through a rotating cylinder and measuring the time required for the molecule to return to its original state after removal of the driving force. The two states, extended and original, in solution would differ in viscosity and the time for the molecule to return to its native state depends on its size. The size of
Botany Plant Molecular Biology Chromosomes
the largest DNA molecule can be estimated by this procedure though there may be shearing of some molecules which is kept to the minimum by isolating DNA molecules by the lysis of the cells right in the chamber of the viscoelastmeter. Using viscoelastometric method the largest DNA molecule of Drosophila was estimated to have a molecular weight of 4.1X1010 daltons which is almost equal to the molecular weight 4.3X1010 daltons estimated by microspectrophotometry.
2.2.3.4 Autoradiography of Drosophila Chromosomes
Autoradiographic studies of pre-labeled DNA of Drosophila, isolated under conditions of least shearing stress and picked up on a membrane, have also yielded results similar to those obtained by viscoelastometry. However, the size of the largest DNA molecule estimated through such studies was 2.4 to 3.2X1010. As the estimated size is almost two-third to three-fourth of the size of DNA present in the largest chromosome of the organism, these results could be taken as supportive of unineme model of chromosome rather than conclusive.
2.2.3.5 Analysis of Yeast Chromosomes
By far the most direct evidence for the existence of a single DNA duplex in each chromosome is provided by the separation of DNAs from the chromosomes of the yeast, Saccharomyces cerviasiae. DNA molecules from each of the small chromosomes (2.3X105 to 1.5X106bp) of the yeast can be extracted, separated and individually identified by pulsed-chase gel electrophoresis.
2.2.3.6 Linkage Analysis
Last but the most compelling indirect evidence for the unineme model of chromosome is that in all organisms the number of linkage groups always equal the number of chromosomes of that organism.
2.2.5 Polytene Chromosomes
The polytene chromosomes provide an exception to the unineme model of chromosomes. These are present in metabolically active organs, such as salivary glands, intestine and excretory organs of dipteran insects, the best example of which being Drosophila melanogaster. The cells of such organs in are almost thousands time larger than the normal cells. These cells contain giant chromosomes, which are several hundred
Botany Plant Molecular Biology Chromosomes
micrometers in length and several micrometers in thickness. They are visible even during interphase stage of cell cycle and exhibit typical banding patterns (Fig. 16). Using cytochemical methods, Hewson Swift (Fig. 17) in 1960s revealed that these, such chromosomes are formed as a result of series of 10 duplications of DNA, none of which is followed by segregation. Thus, these chromosomes contain 1024 DNA molecules arranged parallel tom each other, therefore, they show banding patterns even at interphase stage. These chromosomes provide metabolic advantage to cells because of multiple copies of genes which ensures high level of gene expression.
Fig. 16: Drosophila Polytene Chromosome Fig. 17: Hewson H. Swift
(from staff.jccc.net Control of gene expression) (http://swiftmemorial.uchicago.edu/)
2.2.6 Linear Nature of DNA
One of the distinguishing features between prokaryotic and eukaryotic chromosomes considered to be rule at one time was circular v/s linear nature of DNA in chromosomes, respectively. The evidences that are provided to arrive at the conclusion that eukaryotic chromosomes possess linear DNA molecule are as follows:
2.2.6.1 Linear Genetic Maps
The genetic maps which are based on recombination frequencies between genes on homologous chromosomes of all available chromosomes are linear.
Botany Plant Molecular Biology Chromosomes
2.2.6.2 Translocations are Linear
Translocation is a type of chromosomal rearrangements involving exchange of large pieces of chromosomes between two non-homologous chromosomes. These can be reciprocal or non-reciprocal (fig. 18, 19). Such exchanges can be visualized at light microscopic level. Such exchanges have also been observed to linear.
Fig. 18: Diagrammatic representation of Fig. 19: Diagrammatic representation of reciprocal
Non-reciprocal translocation. Reciprocal translocation
(http://www.emunix.emich.edu/~rwinning/ (http://www.vivo.colostate.edu/hbooks/
genetics/pics/rectrans.gif) genetics/medgen/chromo/recip_xloc.gif)
2.2.6.3 Sister-chromatid Exchanges are Linear
Like reciprocal translocation, sister chromatid exchanges also result in transfer of large pieces of chromatids which are visible at light microscopic of level. However these exchanges take place between sister-chromatids of the same chromosome during mitosis by a process known as mitotic crossing over. These exchanges have always been found to linear and reciprocal. The sister chromatid exchanges in cultured cells can be detected and visualized by a simple experiment. The experiment involves growing of the cells in a medium incorporated with bromodeoxyuridine, a heavy analog of thymidine, for a period equal to one cell cycle, followed by transfer to a normal medium. The chromosomes that divide in the presence of the analog produce daughter chromosomes with DNA molecules having one heavy and other light strand. In the next cycle, when the cell divides in the normal medium, sister chromatid exchanges result in chromosomes parts some of which of which are heavy and others light. These can be visualized by staining chromosomes with Giemsa or a fluorescent stain which impart darker or brighter colour, respectively, to portions containing the analog Fig. 21, 22).
Botany Plant Molecular Biology Chromosomes
Fig. 21: Digramatic representation of sister- Fig. 22: Chromosome exhibiting sister- chromatid exchange. chromatid exchange at metaphase stage.
(http://www.madsci.org/posts/archives/ (http://www.crios.be/genotoxicitytests/
2000-08/967264433.Ge.2.gif) foto_SCE.gif)
